Ischemia is the decrease in blood supply to an organ due to defects in the vasculature carrying blood to such organ. Ischemia results in hypoxia, as oxygen is supplied through organ blood perfusion. The effects of hypoxia may be detrimental to the long-term function of the affected organ, as cell death occurs with sustained hypoxia. Moreover, ischemia may be lethal. Ischemia can result from, for example, cardiovascular disease in stroke and myocardial infarction, acute kidney injury, cerebral trauma, in association with neurodegeneration, as well as from anti-cancer therapy.
Hypoxia is a common etiology of cell injury in human disease, including stroke, myocardial infarction, and solid tumors. Over the past two decades, cell adaptation to hypoxia has emerged as a well-defined active process. Each cell of a multicellular organism can respond to hypoxia by building up hypoxia inducible factor (HIF), a ubiquitous transcription factor capable of activating a battery of genes including genes involved in glucose uptake and metabolism, extracellular pH control, angiogenesis, erythropoiesis, mitogenesis, and apoptosis. The discovery of HIF opened new horizons for the treatment of ischemia and cancer: upregulation of HIF levels has been shown to be beneficial for ischemic diseases, stem cell proliferation [Zhang, C. P., et al., Neurosignals 15, 259-265 (2006)] and transplantation [Liu, X. B., et al., J Cell Biochem 106, 903-911 (2009)], while down-regulation of elevated HIF, a marker of most aggressive cancers, represents a new approach for cancer treatment.
HIF consists of two subunits HIF-1α and HIF-1β, among which HIF-1α is rapidly degraded under normoxic conditions, while HIF-1β is stable [Wang, G. L., et al., Proc Natl Acad Sci USA 92, 5510-5514 (1995); Wang, G. L., et al., J Biol Chem 270, 1230-1237 (1995)]. HIF levels are regulated primarily by post-translational modification of conserved proline residues. Hydroxylation of Pro564 and/or 402 residues in HIF-1α is a prerequisite for its interaction with the von Hippel-Lindau (VHL) protein, yielding a complex that provides HIF ubiquitinylation and subsequent proteasomal degradation [Kaelin, W. G., Jr., Biochem Biophys Res Commun 338, 627-638 (2005)]. Hydroxylation of Pro564 occurs prior to Pro402 [Chan, D. A., et al., Mol. Cell. Biol. 25, 6415-6426 (2005)], although some experiments contradict this finding [Villar, D., et al., Biochem J 408, 231-240 (2007)]. Hydroxylation of HIF-1α Arg803 blocks its interaction with transcriptional proactivator p300 [Lando, D., et al., Science 295, 858-861 (2002)]. In both cases, HIF hydroxylation is executed by α-KG-dependent non-heme iron dioxygenases, HIF prolyl-4-hydroxylase (PHD1-3 isozymes) and asparaginyl hydroxylase (or the so-called FIH, factor inhibiting HIF) [Hirota, K., et al., Biochem Biophys Res Commun 338, 610-616 (2005)].
HIF1 also upregulates a number of prodeath proteins, and thus, HIF1 upregulation can be either prodeath or prosurvival. However, recent evidence [Siddiq, A., et al., J Biol Chem 280, 41732-41743 (2005); Knowles, H. J., et al., Circ Res 95, 162-169 (2004); Baranova, O. et al., J Neurosci 27, 6320-6332 (2007)] strongly suggests that PHDs and FIH are important targets for medical intervention for a number of conditions including chronic anemia and stroke. PHD inhibitors abrogate the ability of HIF1-mediated transactivation of BNIP3 and PUMA to potentiate oxidative death in normoxia [Aminova, L., et al., Antioxidant & Redox Signaling 10, 1989-1998 (2008)]. Although new targets for intervention in the HIF pathway are constantly emerging, the latter observation justifies the search for PHD inhibitors rather than for other types of HIF activators. New substrates have been recently identified for PHD1 (e.g., Rpb1, large subunit of RNA polymerase II [Milchaylova, O., et al., Mol Cell Biol 28, 2701-2717 (2008)] responsible for the fundamental enzymatic activity of the complex, synthesizing all cellular mRNAs) and PHD3 (e.g., β2-adrenergetic receptor [Xie, L., et al., Science Signaling 2, 1-10 (2009)], whose sustained down-regulation is associated with heart failure and asthma) placing HIF PHDs into the focus of drug development efforts. Despite characterization of HIF PHDs as a potential target for anti-ischemic therapy, there has been very little progress in identifying lead compounds that function as HIF PHD inhibitors.